Members of the transforming growth factor-beta (TGF-β) superfamily possess physiologically important growth-regulatory and morphogenetic properties (Kingsley et al., Genes Dev. 8:133-146 (1994); Hoodless et al., Curr. Topics Microbiol. Immunol. 228:235-272 (1998)). Considerable attention has focused in recent years on the role of two TGF-β superfamily members in ovarian function and fertility: growth and differentiation factor-9 (GDF-9) and bone morphogenetic protein-15 (BMP-15, also known as GDF-9b; McNatty et al., Reprod. 128:379-386 (2004); Juengel et al., Hum Reprod. Upd. 11:144-161 (2005)).
GDF-9 was first described in 1993 as a novel member of the TGF-β superfamily which is specifically expressed in the ovary (McPherron et al., J. Biol. Chem. 268:3444-3449 (1993)). Like other members of the TGF-β family, GDF-9 is encoded as a prepropeptide consisting of a signal peptide, a proregion, and a C-terminal mature region which is cleaved from the precursor peptide by an intracellular protease belonging to a group of furin-like proteases. GDF-9 mRNA is present in oocytes at all stages of folliclular development, and is expressed from the primary follicle stage until after ovulation (McPherron and Lee, J. Biol. Chem. 268: 3444-3449 (1993); McGrath et al., Mol. Endocrinol. 9:131-136 (1995); Elvin et al., Mol. Endocrinol. 13:1035-1048 (1999)). Female mice lacking GDF-9 are infertile due to the arrest of developing oocytes at the primary follicle stage (Dong et al., Nature 383:531-535 (1996); Carabatsos et al., Dev. Biol. 204: 373-384 (1998)).
BMP-15, also known as GDF-9b, was discovered as an X-linked gene that encodes a homologue of GDF-9, sharing 52% amino acid identity to GDF-9 in the mature regions (Dube et al., Mol. Endocrinol. 12:1809-1817 (1998); Laitinen et al., Mech. Dev. 78:135-140 (1998)). Although BMP-15 expression is identical to that of GDF-9, it cannot compensate for absence of GDF-9 in a GDF-9 knockout mouse. Female mice lacking BMP-15 are subfertile, demonstrating reduced lifter sizes and litters per month (Yan et al., Mol. Endocrinol. 15: 854-866 (2001)). In sheep, both GDF-9 and BMP-15 are essential for fertility (Juengel et al., Bio. Reprod. 67: 1777-1789 (2002)). Naturally occurring BMP-15 mutations in sheep cause infertility in homozygous females. BMP-15 also plays a critical role in human female fertility, as a BMP-15 mutation has been associated with ovarian dysgenesis in women (Di Pasquale et al., Am. J. Hum. Genet. 75:106-111 (2004)).
GDF-9 and BMP-15 are unique among members of the TGF-β family described to date in that they lack the fourth of seven characteristic conserved cysteine residues in the mature region (Dube et al., Mol. Endocrinol. 12:1809-1817 (1998); Laitinen et al., Mech. Dev. 78:135-140 (1998)). The fourth cysteine is of particular importance because it is responsible for forming the disulfide bond between the subunits of the mature dimer of most TGF-β family members. Although GDF-9 and BMP-15 lack this cysteine and do not form covalently linked dimers, studies have found that both GDF-9 and BMP-15 can form homodimers as well as heterodimers in vitro (Liao et al., J. Biol. Chem. 278:3713-3719 (2003); Liao et al., J. Biol. Chem. 279:17391-17396 (2004)).
Expression of both GDF-9 and BMP-15 is primarily restricted to the oocytes of growing follicles in mammals. Consistent with this expression pattern, no effects outside the ovary have been seen in animals carrying mutations in these genes, including sheep and mice, nor in sheep immunized with GDF-9 or BMP-15 peptides (Galloway et al., Nat. Genet 25:279-283 (2000); Hanrahan et al., Biol. Reprod. 121:843-852 (2004); Dong et al., Nature 383:531-535 (1996); Yan et al., Mol. Enocrinol. 15:854-866 (2001); Juengel et al., Biol. Reprod. 70:557-561 (2004); and Elvin et al., Mol. Endocrinol. 13:1018-1034 (1999)). Thus, these factors have been considered attractive targets for manipulating fertility with a low risk of non-ovarian side effects, including for the purpose of developing new clinical treatments for female infertility, for developing new non-steroidal contraceptives for women, and for modulating fertility in agricultural settings (see, e.g., U.S. Pat. No. 6,030,617).
Although primarily localized to the ovary, GDF-9 and BMP-15 expression have been observed in non-ovarian tissues, including the pituitary and testis (Fitzpatrick et al., Endocrinol. 139:2571-2578 (1998); Aaltonen et al., J. Clin. Endocrinol. Metab. 84:2744-2750 (1999); Eckery et al., Mol. Cell. Endocrinol. 192:115-126 (2002); Otsuka and Shimasaki, Endocrinol. 143:4938-4941 (2002)). However, because expression of these proteins is largely limited to the ovary, non-reproductive functions and uses for GDF-9 and BMP-15 have not received much attention.
A number of conditions are associated with a loss of bone, particularly in the elderly and/or postmenopausal women. For example, osteoporosis is a debilitating disease characterized by a decrease in skeletal bone mass and mineral density, structural deterioration of the bone, and corresponding increases in bone fragility and susceptibility to fracture. Osteoporosis in humans is preceded by clinical osteopenia, a condition found in approximately 25 million people in the United States.
Throughout adult life, bone continually undergoes a turnover through the coupled processes of bone formation and resorption. Bone resorption is mediated by bone resorbing cells, osteoclasts, which are formed by mononuclear phagocytic cells. New bone replacing the lost bone is deposited by bone-forming cells, osteoblasts, which are formed by mesenchymal stromal cells. Various other cell types that participate in the remodeling process are tightly controlled by systemic factors (e.g., hormones, lymphokines, growth factors, and vitamins) and local factors (e.g., cytokines, adhesion molecules, lymphokines, and growth factors). The proper spatiotemporal coordination of the bone remodeling process is essential to the maintenance of bone mass and integrity. A number of bone degenerative disorders are linked to an imbalance in the bone remodeling cycle which results in abnormal loss of bone mass (osteopenia) including metabolic bone diseases, such as osteoporosis, osteoplasia (osteomalacia), osteodystrophy, and Paget's disease.
There are currently two main types of pharmaceutical therapy available for the treatment of osteoporosis. The first, and most common, approach is the use of hormone therapy to reduce the resorption of bone tissue. Estrogen replacement therapy (“ERT”) is known to prevent further deterioration and thus reduce the likelihood of fractures. However, the use of estrogen as a treatment is limited, as it is believed that long-term estrogen therapy may be associated with risk of uterine cancer, endometrial cancer, breast cancer, frequent vaginal bleeding, and thrombosis. Because of these serious side effects, many women choose to avoid this treatment. Further, few men agree to this type of therapy. The second major therapeutic approach to osteoporosis is the use of bisphosphonates, particularly alendronate, risedronate, and ibandronate. Although tests have shown that these compounds consistently increase the bone mineral density in osteoporosis patients, there are also significant problems with the treatment of osteoporosis by bisphosphonates, including irritation of the esophagus and upper gastrointestinal tract.
Therefore, there exists a need to develop new therapeutic methods for treating and preventing bone disorders.